Prior Art Statement
The possibility of transferring specific functional genes from one species to another has intrigued scientists for years. However, such transfers have only been realized in cell culture systems. Cloned rabbit .beta.-globin gene sequences have been stably introduced into thymidine kinase (TK) deficient mutant mouse cells by DNA mediated cotransformation using the rabbit .beta.-globin gene and herpes virus TK gene sequences, Wigler, M., et al, Cell, 11: 223-232 (1977). This approach has been extended to use cellular TK, Wigler, M., et al, Cell, 14: 725-731 (1978), adenine phosphoribosyl transferase, Wigler, M., et al, Proc. Natl. Acad. Sci. U.S.A. 76: 1373-1376 (1979), and hypoxanthine phosphoribosyl transferase, Graf, L. H., et al., Som. Cell Genet., 5: 1031-1044 (1979), genes as unlinked but selectable markers.
Additionally, the use of a mutant hamster gene, which codes for an altered dihydrofolate reductaase, as a selectable marker offering methotrexate resistance, allows the introduction and amplification of a broad range of genetic elements into a variety of cell lines, Wigler, M. et al; Proc. Natl. Acad. Sci. U.S.A., 77: 3567-3570 (1980). Although these cotransformation experiments enable a direct gene transfer between mammalian species to be effected, these techniques are all restricted to cell culture systems wherein only one cell in 10.sup.5 -10.sup.7 is transformed which requires that the transformed cells be selected for on restrictive media.
The direct microinjection of DNA has been used to introduce Herpes Simplex virus TK gene into cultured mammalian cells. Capecchi, M., Cell, 22: 479-488 (1980). However, the efficiency of transformation was extremely low even when the small pBR 322/TK DNA was coinjected with SV40 DNA, i.e., 15 transformants per 1,000 cells injected. Additionally, this technique does not result in the production of a mature organism much less one that could exhibit a phenotypic alteration.
Mosaic mice have been constructed by the injection of tetracarcinoma cells into the blastocysts of developing mice. Brinster, R. L., J. Exp. Med., 140: 1049-1056 (1974) Mintz, B., et al, Proc. Natl. Acad. Sci. U.S.A., 72: 3585-3589 (1975) and Papaioannou, V. E., et al, Nature, 258: 69-73 (1975). Teratocarcinoma cells have also been used as vehicles for introducing genes into mice to produce mosaic mice. Pellicer, A., et al, Proc. Natl. Acad. Sci. U.S.A., 77: 2098-2102 (1980), Watanabe, T., et al, Proc. Natl. Acad. Sci. U.S.A., 75: 5113-5117 (1978) and Illmensee, K., et al Proc. Natl. Acad. Sci. U.S.A., 75: 1914-1981 (1978). However, by definition, a mosaic mouse has patches of different, mutually exclusive genotypes and/or phenotypes. Additionally, the possibility of germ-line transmission with mosaic mice is reduced due to the fact that teratoma cells of XX chromosomal constitution cannot make sperm in mice that develop as males.
Recently, Gordon, et al, Proc. Natl. Acad. Sci. U.S.A., 77: 7380-7384 (1980), have observed TK genes in mice developed from embyros microinjected with the TK gene incorporated into a chimeric SV40 viral vehicle. These mice, however, did not express the TK gene product. In these experiments, as well as the experiments relating to the use of tetratocarcinoma cells, no phenotypic alteration of the test animal was accomplished. The results reported by Gordon, et al, are not surprising since the SV40 virus has previously been shown to be functionally and genetically inactive when introduced into mouse embryos. Jaenisch, R. et al, Proc. Natl. Acad. Sci. U.S.A., 71: 1250-1254 (1974).
Disclosure of the Invention:
Genetic transformation of a zygote and the embryo and mature organism which result therefrom is obtained by the placing or insertion of exogenous genetic material into the nucleus of the zygote or to any nucleic genetic material which ultimately forms a part of the nucleus of the zygote. The genotype of the zygote and the organism which results from the zygote will include the geotype of the exogenous genetic material. Additionally, the inclusion of the exogenous genetic material in the zygote will result in a phenotypic expression of the exogenous genetic material. The genotype of the exogenous genetic material is expressed upon the cellular division of the zygote. However, the phenotypic expression, e.g., the production of a protein product or products of the exogenous genetic material or alteration of the zygote's or organism's natural phenotype, will occur at that point of the zygote or organism's development during which the particular exogenous genetic material is active. Alteration of the expression of a phenotype includes an enhancement or diminishment in the expression of a phenotype or an alteration in the promotion and/or control of a phenotype including the addition of a new promotor and/or controller or supplementation of an existing promoter and/or controller of a phenotype.
The present invention has application in the genetic transformation of multicellular eucaryotic organisms which undergo syngamy, i.e., sexual reproduction by the union of gamete cells. Examples of such organisms include amphibians, reptiles, birds, mammals, bony fishes, cartilaginous fishes, cyclostomes, arthropods, insects, mollusks, thallophytes, embryophytes including gymnosperms and angiosperms. Preferred organisms include mammals, birds, fishes, gymnosperms and angiosperms.
The invention is particularly useful in the breeding of plants and animals, especially ones of agricultural value, to obtain species having a genetic makeup which results in a plant or animal having more desirable characteristics. Since the source of the exogenous genetic material can be from animals or plants, synthetic equivalents of naturally occurring genetic material or totally new synthetically produced genetic material and from the same or a different species of the zygote being transformed, the invention can be used to modify a species or create a new species. Modification of a species is obtained when the genotype of the exogenous genetic material occurs in the genotype of the species whose zygote is being genetically transformed. A new species is obtained when the genotype of the exogenous genetic material occurs in another species and does not naturally occur in the species of the zygote being genetically transformed. For example, increased growth rate and the efficiency of feed utilization can be obtained by genetic transformation of animals used to produce meat. As an example, the genes relating to growth rate and feed utilization can be transferred from a buffalo into beef cattle which would create a new species. Dairy animals can undergo an increase in milk production and efficiency of feed utilization by transferring exogenous genetic material from species or breeds of the same species which have either or both traits. The quality and flavor of meat, for example, lamb, can also be enhanced in a similar manner. Additionally, the invention can be used as an in vivo analysis of gene expression during differentiation and in the elimination or dimunition of genetic diseases, e.g., hemophilia, Tay-Sachs disease, phenylketonuria, homocystinurea, galactosemia, thalassemia and sickle cell anemia.